Multigene typing of Giardia Duodenalis isolated from tuberculosis and non-tuberculosis subjects

Giardia duodenalis is a cryptic protozoan, which has eight assemblages (A-H). Assemblages A and B are the main genotypes reported from humans with probable anthroponotic and zoonotic transmission. The current study aimed to characterize G. duodenalis assemblages in tuberculosis (TB) patients and healthy subjects using multilocus genotyping (MLG). Thirty Giardia-positive stool samples, which were obtained from TB patients and healthy subjects were included in the study. After total DNA extraction, three β-giardin (bg), triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) genes were amplified and sequenced. Obtained sequences were compared to the GenBank database to characterize assemblages. Phylogenetic analysis using Maximum Likelihood (ML) and Tamura 3-parameter was performed for each gene. From 30 Giardia-positive subjects, 17 (57%) and 13 (43%) were from healthy and TB-infected subjects, respectively. There was no significant co-existence of Giardia and tuberculosis (P-value = 0.051). In addition, 14 (46.7%) and 16 (53.3%) of Giardia isolates were from asymptomatic and symptomatic subjects, respectively. PCR amplification was successful in 25 single samples (83.3%) consisted of 20 for tpi, 15 for bg, and 13 for gdh genes. Accordingly, 13/25 (52%) and 8/25 (32%) belonged to assemblage A and assemblages B, respectively, whereas 4/25 (16%) were either assemblage A or B with different genes at the same time. Significant correlation between assemblages and TB, age, and symptoms was not seen. The phylogenetic analyses represented no separation based on TB and gastrointestinal symptoms. Assemblage A was the predominant genotype in samples. The high frequency of assemblage AII indicated importance of anthroponotic transmission of Giardia in both healthy and TB patients. In addition, considering the exclusive reports of sub-assemblage AIII in wild ruminants, the presence of AIII in the current study have to be carefully interpreted. The inconsistency between the assemblage results of either bg or gdh loci with tpi gene signifies the insufficiency of single gene analysis and the necessity for MLG in molecular epidemiology of G. duodenalis.


Introduction
Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) is a cosmopolitan flagellated protozoan, which is reported from not only humans, but also a broad spectrum of animals. In addition to G. duodenalis, there are at least seven other species including G. agilis, G. ardeae, G. psittaci, G. muris, G. microti, G. peramelis, and G. cricetidarum, which colonize the intestine of a broad variety of animals from amphibians to birds [1]. Unlike other species, G. duodenalis is not limited to a specific host, and is reported from humans and domesticated/wild animals, which increases the probability of zoonotic transmission [2]. The main transmission rout of Giardia spp., in humans is fecal-oral via ingestion of infective cysts defecated by either humans or animals, as well as contaminated food and water [3][4][5].
Although infection by G. duodenalis in humans is mostly asymptomatic, variety of gastrointestinal manifestations such as bloating, nausea, flatulence, fatigue, weight loss, diarrhea, and steatorrhea might be reported [6][7][8]. The correlation between diarrhea, as the main symptom of giardiasis, and colonization of Giardia has been controversial [9,10]. However, it is suggested that the presence of G. duodenalis may protect infected subjects from diarrhea due to other intestinal pathogens [6].
Despite the similar morphology, eight assemblages (A-H) has been characterized based on the molecular diversity within G. duodenalis genomes. Accordingly, assemblages A and B are the major reported genetic lineages from humans and animals, while other six assemblages are exclusively reported from animals [11,12]. Nevertheless, the majority of assemblages A and B in humans and high occurrence of host-adapted assemblages in companion animals suggest that the zoonotic transmission of G. duodenalis is less common than expected before [11].
The presence and assemblage distribution of G. duodenalis have been evaluated among different groups of healthy subjects and persons with background diseases. However, there are limited data about the assemblages of G. duodenalis in patients who suffer from tuberculosis (TB) [16][17][18][19]. Indeed, there is no data about distribution of assemblages in TB patients. Therefore, the current study aimed to characterize assemblages of G. duodenalis isolated from human subjects with and without TB. Consent was informed to participates and verbal consent was obtained from all subjects and/or their legal guardian(s). For those patients with age�16, informed consent was obtained from their respective parent(s)/guardian(s) as well.

Stool sample collection and DNA extraction
This study was conducted on 30 Giardia-positive samples, which had been obtained from 427 stool samples collected from our previous studies (261 TB patients) [19,20], as well as samples, which were referred to the Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences. All samples were either positive or suspected for G. duodenalis using lugol's iodine staining and microscopically examination. Total DNA was extracted from stool samples using DNA stool extraction mini kit (Yekta Tajhiz, Tehran, Iran) with some modifications [21]. Purified DNA was stored at -20˚C.

Multilocus genotyping
Nested PCR was employed to amplify the bg, gdh, and tpi genes among G. duodenalis isolates using primers and PCR conditions, which were mentioned elsewhere [22] (Table 1). To characterize assemblages of each isolate, PCR products were sequenced. Raw sequence data in forward direction was viewed using the Chromas Lite version 2.6 sequence analysis program (https://chromas.software.informer.com/2.6/). The nucleotides were checked and manually edited, where required. The BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare nucleotide sequences with sequences previously submitted to the GenBank database.

Phylogenetic analyses
Generated sequences were trimmed and aligned based on appropriate reference sequences using BioEditv.7.2.6 software. Phylogenetic trees were constructed for targeted fragments of each tpi, bg, and gdh gene of G. duodenalis using the Maximum-likelihood algorithm and Tamura 3-parameter model in MEGAX software, together with a number of sequences, which were retrieved from the GenBank database to evaluate the molecular distance and phylogenetic GiarR CTCGACGAGCTTCGTGTT � Primers used in the first PCR step.Edited sequences, and their resulting amino acid sequences, were submitted to the GenBank database under accession numbers: OM115964 to OM115983, OM115984 to OM115998, and OM115999 to OM116011, for the tpi, bg, and gdh loci, respectively.

Statistical analysis
Statistical analyses, Pearson's Chi-square (χ2) for independence and Fisher's exact tests incorporated in SPSS version 23 software (SPSS Inc. Chicago, IL, USA) were employed to compare the frequency of G. duodenalis assemblages in symptomatic and asymptomatic subjects. Statistical significance was set as a P-value < 0.05.
From Giardia-positive samples, 17 (57%) were males and 13 (43%) were females. There was no statistically significant correlation between the presence of Giardia and gender (P-value = 0.709).
Among TB patients, from 13 Giardia-positive samples, assemblages A and B were characterized among seven (53.84%) and three (23.07%), respectively, while three (23.07%) of remained samples did not amplified or were failed in sequencing. From 17 Giardia-positive samples, Assemblages A and B were characterized among six (35.29%) and five (29.41%) of non-TB subjects, respectively, while four (23.53%) samples were assemblage A or B with different genes and two (23.07%) did not amplified or were failed in sequencing.

Phylogenetic analyses
Phylogenetic analysis of bg, tpi, and gdh genes revealed that assemblages A and B were clearly separated and grouped with reference assemblages retrieved from the GenBank database for each gene (Fig 1). Moreover, the phylogenetic analysis of sequences of all three genes represented that there was no separation based on the presence of TB, gastrointestinal symptoms, sources, and geographical areas.

Discussion
Giardia duodenalis is a prevalent protozoan, particularly in developing region, which infects humans and a board range of animals [13]. The prevalence of G. duodenalis in Iran has been even reported more than 30%; however, the prevalence rate may vary regarding the studied population and employed diagnostic methods [24][25][26][27][28]. Although among epidemiological surveys, G. duodenalis is one of the most frequently reported protozoan, our knowledge is still insufficient about the molecular epidemiology and circulating assemblages of the parasite in Iran. The current study is one of the rare research mining the molecular characterization and assemblages of G. duodenalis in TB patients using multilocus genotyping (MLG) in the world. Assemblage characterization of G. duodenalis is a challenge, since multi-copy genes, like ssu rRNA gene, are not discriminative enough to identify assemblages and sub-assemblages [29,30]. Besides, success rate for amplification of single copy genes, which are discriminative, varies from 11 to 90% [31]. Therefore, multigene typing is a valid model for molecular characterization of G. duodenalis at assemblage and sub-assemblage level [13]. Among reliable genes for assemblage characterization of G. duodenalis, three loci tpi, gdh, and bg are well-known genetic targets [13]. In the current study, from 30 microscopically positive samples, 25 samples were amplified by each/two/all genes including 20, 15, and 13 samples for tpi, bg, and gdh, respectively. This observation is in accordance to previously published papers indicating inconsistency between the prevalence of G. duodenalis-positive samples in microscopy and molecular amplification [32][33][34]. This discrepancy could be related to the quality of extracted DNA, the copy number of targeted genes, and the presence of PCR inhibitors [31,34].
In the current study, three and seven sequences of bg and gdh genes were highly similar to bacteria (Bifidobacterium, Feacalibacterium, Pseudomonas, Kinneretia and Escherichia coli) and virus (Siphoviridae), while none of tpi locus sequences were identical to non-Giardia sequences. Such results highlight the concern for false positive results of non-specific target gene amplification by PCR without sequencing [31].
As a result, assemblage A was the most prevalent genotypes in the current study. Assemblage A is the predominant genetic lineage of G. duodenalis in humans in most of molecular  studies in Iran [28,34,35]. In a study conducted by Sarkari et al. [36], assemblage A was identified among 74.4% of Giardia-positive samples based on the amplification followed by restriction fragment length polymorphism (RFLP) of partial fragment of gdh gene. Mahmoudi et al. [28] reported assemblage A as the major genotype based on the partial amplification and sequencing of gdh gene. In addition, Bahramdoust et al., [35] designed and evaluated a realtime PCR coupled with high resolution melting curve analysis (HRM) to detect and characterize G. duodenalis in humans and dogs, and reported assemblage A as the major genotype in humans. Although assemblage A seems to be the predominant genotype in humans in Iran [37][38][39], there are controversial results, as well. For example, in a MLG study conducted in Iran, the presence of assemblages A and B were similar [34]. Most of assemblage A and all of assemblage B were AII and BIII/BIV, respectively, which are supposed to be responsible for anthroponotic transmission [2,34]. Interestingly, an inconsistency between AII and AIII was observed in sub-assemblage analysis by different genes. In addition, all sub-assemblage AIII were identified based on bg gene sequencing. However, AIII was supposed to be exclusively reported from wild ruminants [40,41], and the presence of this sub-assemblage in this study may be attributed to the misassigned sequences, which have been submitting to the GenBank database, and have to be carefully interpreted. Furthermore, it is suggested to employ either/ both tpi or/and gdh genes alongside with bg locus for sub-assemblage analysis. Regarding our results, significant correlation was not seen between clinical symptoms and certain assemblage. Furthermore, there was no correlation between certain assemblage and tuberculosis. Actually, a little is known about the correlation between genetic variability of Giardia and presentation of clinical symptoms. Although there are reports demonstrating an association between assemblages and type of symptoms [42,43], most of studies failed to link genetic variability of Giardia with symptoms [34,44,45].
Phylogenetic analysis showed that all sequences were clearly separated based on the assigned assemblages and grouped with their reference sequences for each gene. However, an inconstancy was appeared between the assigned assemblages by either bg or gdh genes and tpi. In another word, in four G. duodenalis sequences, which were characterized as assemblages either A or B with two bg and gdh genes, the results of tpi gene was conflicting. This issue could be related to the segregation sites, the number of mutation, single nucleotide polymorphisms (SNPs), and discriminatory power of each target gene [13]. This finding highlights the insufficiency of a single target gene screening and the need for MLG investigation for molecular epidemiology studies of Giardia.

Conclusion
This study is the first analyzing G. duodenalis assemblages in TB patients using MLG approach. The assemblage A was the predominant genotype in our isolates, but a significant correlation between certain assemblage with symptoms and TB was not observed. The high prevalence of assemblage AII indicated the importance of anthroponotic transmission of Giardia in both healthy and TB-infected subjects. In addition, considering the exclusive reports of sub-assemblage AIII in wild ruminants, the presence of AIII in the current study have to be carefully interpreted. The inconsistency between the assemblage results of either bg or gdh genes and tpi gene signify the insufficiency of single gene analysis and the necessity of MLG in molecular epidemiology of G. duodenalis.